Carbon-Fiber Nanoelectrodes for Real-Time Discrimination of Vesicle Cargo in the Native Cellular Environment.
Carbon-Fiber Nanoelectrodes for Real-Time Discrimination of Vesicle Cargo in the Native Cellular Environment.
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DOI：
10.1021/acsnano.9b07318
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发表时间：
2020-02
期刊：
影响因子：
17.1
通讯作者：
James G. Roberts;Edwin C Mitchell;Lars E. Dunaway;G. McCarty;Leslie A. Sombers
中科院分区：
文献类型：
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作者：
James G. Roberts;Edwin C Mitchell;Lars E. Dunaway;G. McCarty;Leslie A. Sombers
碳纤维微电极已被证明是一个不可或缺的工具，用于监测胞吐事件使用安培法。当定位在细胞附近时，传统的微盘电极非常适合于量化离散的胞吐释放事件。然而，电极的尺寸不允许细胞内电化学测量，并且安培法无法区分释放的儿茶酚胺。在这项工作中，开发了碳纳米电极，允许选择性电化学采样的细胞胞质溶胶中的纳米囊泡。经典的伏安技术和电子显微镜被用来表征纳米电极，这些纳米电极长约5微米，并被削尖成纳米级的尖端，可以完全插入单个神经内分泌细胞。将纳米电极与快速扫描循环伏安法（FSCV）相结合，以区分含有肾上腺素的分泌颗粒与在天然细胞环境中遇到的其他含有儿茶酚胺的颗粒。这两种囊泡亚型中遇到的大多数细胞，尽管先前的证明群体的嗜铬细胞，优先释放这些儿茶酚胺之一。相对肾上腺素含量在细胞与细胞之间有很大的差异，含有肾上腺素的囊泡通常比其他囊泡储存更多的儿茶酚胺。因此，碳纳米电极技术能够分析皮升规模的生物体积，揭示了嗜铬细胞在致密核心颗粒水平上的关键差异。
Carbon-fiber microelectrodes have proven to be an indispensable tool for monitoring exocytosis events using amperometry. When positioned adjacent to a cell, a traditional microdisc electrode is well suited for quantification of discrete exocytotic release events. However, the size of the electrode does not allow for intracellular electrochemical measurements, and the amperometric approach cannot distinguish between the catecholamines that are released. In this work, carbon nanoelectrodes were developed to permit selective electrochemical sampling of nanoscale vesicles in the cell cytosol. Classical voltammetric techniques and electron microscopy were used to characterize the nanoelectrodes, which were ~5 microns long and sharpened to a nanometer-scale tip that could be wholly inserted into individual neuroendocrine cells. The nanoelectrodes were coupled with fast-scan cyclic voltammetry (FSCV) to distinguish secretory granules containing epinephrine from other catecholamine-containing granules encountered in the native cellular environment. Both vesicle subtypes were encountered in most cells, despite prior demonstration of populations of chromaffin cells that preferentially release one of these catecholamines. There was substantial cell-to-cell variability in relative epinephrine content, and vesicles containing epinephrine generally stored more catecholamine than the other vesicles. The carbon nanoelectrode technology thus enabled analysis of picoliter-scale biological volumes, revealing key differences between chromaffin cells at the level of the dense-core granule.